1. Field of the Invention
The present invention relates generally to synthetic biologically active compositions which have a microparticulate (nanoparticulate) core. More particularly, the present invention relates to biologically active compositions where transfecting DNA or RNA is attached to a microparticulate core and coated with a targeting membrane or ligand. These transfection nanoparticles are useful in delivering the transfecting DNA or RNA to target cells.
2. Description of Related Art
The attachment of biologically active proteins, peptides or pharmacologic agents to various carrier particles has been an area of intense investigation. These conjugated biological systems offer the promise of reduced toxicity, increased efficacy and lowered cost of biologically active agents. As a result, many different carrier models are presently available. (Varga, J. M., Asato, N., in Goldberg, E. P. (ed.): Polymers in Biology and Medicine. New York, Wiley, 2, 73-88 (1983). Ranney, D. F., Huffaker, H. H., in Juliano, R. L. (ed.): Biological Approaches to the Delivery of Drugs, Ann. New York Acad. Sci., 507, 104-119 (1987).) Nanocrystalline and micron sized inorganic substrates are the most common carriers and proteins are the most commonly conjugated agents. For example, gold/protein (principally immunoglobulin) conjugates measuring as small as 5 nm have been used in immunological labeling applications in light, transmission electron and scanning electron microscopy as well as immunoblotting. (Faulk, W., Taylor, G., Immunochemistry 8, 1081-1083 (1971). Hainfeld, J. F., Nature 333, 281-282 (1988).)
Silanized iron oxide protein conjugates (again principally antibodies) generally measuring between 500 and 1500 nm have proven useful in various in vitro applications where paramagnetic properties can be used advantageously. (Research Products Catalog, Advanced Magnetics, Inc., Cambridge, Mass., 1988-1989.) Ugelstad and others have produced gamma iron oxides cores coated with a thin polystyrene shell. (Nustad, K., Johansen, L., Schmid, R., Ugelstad, J., Ellengsen, T., Berge, A.: Covalent coupling of proteins to monodisperse particles. Preparation of solid phase second antibody. Agents Actions 1982; 9:207-212 (id. no. 60).) The resulting 4500 nm beads demonstrated both the adsorption capabilities of polystyrene latex beads as well as the relatively novel benefit of paramagnetism.
Carrier systems designed for in vivo applications have been fabricated from both inorganic and organic cores. For example, Davis and Illum developed a 60 nm system comprised of polystyrene cores with the block copolymer poloxamer, polyoxyethylene and polyoxypropylene, outer coats that showed a remarkable ability to bypass rat liver and splenic macrophages. (Davis, S. S., Illum, L., Biomaterials 9, 111-115 (1988)). Drug delivery with these particles has not yet been demonstrated. Ranney and Huffaker described an iron-oxide/albumin/drug system that yielded 350-1600 nm paramagnetic drug carriers. (Ranney, D. F., Huffaker, H. H., In, Juliano, R. L. (ed.): Biological approaches to the delivery of drugs, Ann. New York Acad. Sci. 507, 104-119 (1987).) Poznasky has developed an enzyme-albumin conjugate system that appears to decrease the sensitivity of the product to biodegradation while masking the apparent antigenicity of the native enzyme. (Poznasky, M. J.: Targeting enzyme albumin conjugates. Examining the magic bullet. In, Juliano, R. L. (ed.): Biological approaches to the delivery of drugs, Annals New York Academy Sciences 1987; 507-211:219.)
Shaw and others have prepared and characterized lipoprotein/drug complexes. (Shaw, J. M., Shaw, K. V., Yanovich, S., Iwanik, M., Futch, W. S., Rosowsky, A., Schook, L. B.: Delivery of lipophilic drugs using lipoproteins. In, Juliano, R. L.(ed.): Biological approaches to the delivery of drugs, Annals New York Academy Sciences 1987; 507:252-271.) Lipophilic drugs are relatively stable in these carriers and cell interactions do occur although little detail is known.
In any conjugated biological composition, it is important that the conformational integrity and biological activity of the adsorbed proteins or other biological agents be preserved without evoking an untoward immunological response. Spacial orientation and structural configuration are known to play a role in determining the biological activity of many peptides, proteins and pharmacological agents. Changes in the structural configuration of these compounds may result in partial or total loss of biological activity. Changes in configuration may be caused by changing the environment surrounding the biologically active compound or agent. For example, pharmacologic agents which exhibit in vitro activity may not exhibit in vivo activity owing to the loss of the molecular configuration formerly determined in part by the in vitro environment. Further, the size and associated ability of the carrier particle to minimize phagocytic trapping is a primary concern when the composition is to be used in vivo. All of these factors must be taken into account when preparing a carrier particle.
To date, gene therapy in humans has been limited to ex-vivo protocols in which tissues are transfected in the culture dish and placed back in the body. In vivo work is still in pre-clinical development and has been confined to animal models due to a range of safety and efficacy issues. Such concerns arise primarily from the use of viral vectors to effect the gene transfer. Retroviral transfection has generated a lot of interest since it can stably transfect nearly 100% of targeted cells ex-vivo. Production of transfecting, replication defective retroviruses, proceeds through packaging cell lines which in principle are unable to produce wild type virus. However, low titers of "wild type" (replication competent) virus have been observed in these systems. In one such protocol utilizing primates, outbreaks of lymphoma were linked to the detection of wild type retrovirus from a packaging system. Besides potential pathogenicity, maintaining useful transfecting titers of these vectors can be difficult. They are hard to purify and concentrate since the envelopes (membranes) tend to be extremely labile. Alternatively, adenoviral vectors have been found to be considerably more stable. Moreover, these viruses are capable of transfecting quiescent tissue and producing large amounts of gene products. Unfortunately, gene expression is often transient because the viral genome often remains extrachromosomal. Direct clinical application is also problematic, since replication of the vector can result in aberrant host protein synthesis leading to deleterious effects ranging from oncogenesis to cellular toxicity.
Given the practical concerns of in vivo viral transfection, nonviral methods are also being developed. At present, most efforts are centered on receptor mediated transfer because such methods can provide targeted delivery of DNA (and RNA) in vivo. Receptor-mediated systems employ ligand DNA (and RNA) complexes which can be recognized by cell receptors on the cell surface. Internalization of the complex occurs via the formation of endocytotic vesicles which allow for transport into the cytoplasm. Problems arise, however, when the endosomes fuse with lysosomes which causes the contents to be destroyed. In turn, the transfection rate for these complexes remain below clinical efficacy. Some investigators have used fusogenic peptides of Influenza Hemmoglutin A to disrupt endosome formation which has led to higher transfection rates. Nonetheless, the data on in vivo expression suggests that this method may only permit transient expression of genes.
Besides ligand DNA (and RNA) complexes, lipofection techniques have also been tried with varying success. Liposomes are specially susceptible to uptake by the filter organs and in the peripheral tissues by macrophages which limits their transfection efficiency and specificity in vivo.
In view of the above, it would be desirable to provide compositions which can be used to transfect cells with DNA or RNA in both in vivo or in vitro environments.